Tamoxifen is a mainstay in the treatment of estrogen receptor (ER)-positive breast cancer. However, long-term treatment of women at high risk for breast cancer reduces the incidence of both invasive and non-invasive ER+ breast cancer by only about 50 percent and also modestly increases the risk of endometrial cancer. Breast tumors could also become dependent on the drug for growth. Therefore, there is considerable room for improvement over tamoxifen. The clinical limitation of tamoxifen is reflected in breast and endometrial tumor xenograft models. Understanding different molecular mechanistic aspects of tamoxifen is necessary for improving upon this drug in decreasing the incidence and recurrence of breast tumors. A remarkably under-investigated aspect of estrogen (E2) action is gene repression which is frequently prevented by tamoxifen (de-repression). We have established a novel non-classical mechanism for direct gene repression by E2 in which ER forms a TAFII30-associated co-repressor complex; tamoxifen prevents this repression by simply dissociating the complex. We have identified synthetic ER ligands that mimic tamoxifen in a variety of model promoters and genes; however, these compounds differ from tamoxifen in that they behave like E2 with respect to the above mechanism of direct gene repression. Remarkably, the new compounds not only blocked E2-stimulated MCF-7 breast tumor cell proliferation but were also antiproliferative in tamoxifen-resistant MCF-7 cells developed in vivo; they also blocked E2-stimulated growth of Ishikawa uterine endometrial cancer cells in contrast to tamoxifen. The compounds did not affect the growth of ER-negative cells. The findings beg the question of what the physiological correlates of different mechanisms of gene de- repression by tamoxifen might be, particularly in relation to the effects of tamoxifen on the incidence and growth of breast and endometrial tumors. We hypothesize that gene de-repression by tamoxifen offers distinctive mechanisms that can provide the basis for identification of mechanistic classes of ER antagonists with superior therapeutic effects in breast cancer. In other words, tamoxifen's property of de-repressing certain genes may, in itself or by association with other effects, help in creating a condition that would be permissive to the onset or success of different survival/proliferation mechanisms, thereby constraining the drug's beneficial effects in certain cell contexts; the abrogation of such a mechanism(s) in certain ER antagonists may have a net effect of indirectly suppressing cell survival/proliferation, thereby increasing the duration of tumor suppression. The ER antagonists that we have identified (as well as those we expect to identify in the future) are excellent tools to address the hypothesis since they share a common mechanistic difference related to gene repression/de- repression. Since the expected differences in gene de-repression profiles of the compounds vs. tamoxifen are relatively small, it may even be feasible to use bio-statistical analysis coupled with multiple gene knockdown approaches to ultimately relate a group of E2 targets de-repressed by tamoxifen to specific physiological effects of the drug. Regardless of the outcome, it is necessary and feasible at this time to initiate systematic studies to shed light on the nature and extent of the possible impacts of novel and uninvestigated aspects of the genomic action of tamoxifen. The mechanism-based chemical biology approach proposed here is timely because of the availability of a large collection of partially characterized ER ligands, whose classification has been limited and largely empiric. Aim 1: Further investigate molecular mechanisms of gene de-repression by tamoxifen and identify additional model genes to represent each mechanism. Aim 2: Continue to identify new mechanistic classes of ER antagonists; compare tamoxifen and the new compounds with respect to cellular effects in ER+ breast and endometrial cancer cell lines in vitro; determine differential gene repression patterns and attempt to identify critical gene targets. Aim 3: Undertake a comparative evaluation of the effects of tamoxifen and selected new reagents in relation to inhibition and latency of breast and endometrial tumors using ER+ human tumor cells in xenograft models that have previously provided the most predictive clinical information for tamoxifen and also examine uterotropic effects.